CN108064309A - A kind of method of Enzyme catalyzed synthesis ginseng saponin Rh 2 - Google Patents
A kind of method of Enzyme catalyzed synthesis ginseng saponin Rh 2 Download PDFInfo
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Abstract
The invention discloses a kind of method of Enzyme catalyzed synthesis ginseng saponin Rh 2, this method carries out catalytic reaction from the glucuroide of Terrabacter ginsenosidimutans as substrate addition using ginseng sapoglycoside Rg 3 and is generated as ginseng saponin Rh 2；The temperature of catalytic reaction is 25~50 DEG C；PH value is 6.5~9.5.The method of present invention production ginseng saponin Rh 2 is simple, and reaction selectivity is high, and by-product is few, and glucuroide and its mutant of the present invention are easy to get through Escherichia coli fermentation, and production cost and product quality are suitble to industrialized production better than chemical method.Reaction condition is mild, belongs to environmentally friendly production technology, meets the environmentally protective industrial standard of country's popularization.
Description
Technical field
The invention belongs to native compounds to synthesize field, and in particular to a kind of method of Enzyme catalyzed synthesis ginseng saponin Rh 2.
Background technology
Ginseng is with a long history, as a kind of traditional rare Chinese medicine, has excellent medical value.Ginseng main active
For ginsenoside, including three types, it is respectively：Protopanoxadiol saponins (Protopanaxadiol type
Ginsenoside, PPD), protopanaxatriol saponins (Protopanaxatriol type ginsenoside, PPT) and people
Join saponin(e Ro.Most saponin(es are Rb1, Rb2, Rc, Rd, Re and Rg1 ginsenoside in ginseng.Wherein rare ginsenoside
Rh2 is few in nature content.Ginseng saponin Rh 2 is the natural plant composition that a kind of newly discovered antitumor, anti-rotation are moved, and is
The choice drug of Combined with Radiotherapy, chemotherapeutic sensitivity attenuation, is received by the market as new type anticancer body-building health product.
The chemical structure of general formula of ginsenoside is：
Its species and structure feature are as shown in table 1.
The species and structure feature of 1 ginsenoside of table
Note：Glu：Glucose；Araf：Arabinofuranose；Arap：Arabopyranose；Rha：Rhamnose
Many methods for preparing ginseng saponin Rh 2 exist in the prior art, as CN102352402 B are disclosed from ginseng root
The extract containing ginseng itself saponin(e enzyme and other water-soluble substanceses is extracted, joining the reaction of di-alcohols saponin(e with protoplast prepares
Rh2 mixing saponin(es.101565694 A of CN disclose higher for activity using panax ginsenoside enzymatic conversion notoginsenoside
The secondary saponin(e of Rg3, CK, Rh2 and aglycon.CN 101385519A are disclosed to be generated using a kind of high corn bud of saponin(e enzymatic activity
Enzyme solution, mixed with ginseng pulverate or general ginsenoside or ginsenoside Rb1,40-50 DEG C be stirred to react 10-24 it is small when, generate
Containing ginsenosides such as F2, Rg3, CK, Rh2.Also some methods need to be reacted using full cell, be unfavorable for the abundant of enzyme and substrate
Contact so as to influence conversion ratio, causes reaction selectivity low, Rh2 low outputs, purity are low；Such as CN 1105781C patents, profit
A small amount of ginseng saponin Rh 2 is obtained with beta-glucosidase enzyme hydrolysis ginsenoside, conversion ratio is low, causes reaction selectivity low, Rh2
Low output, purity are low.As it can be seen that the preparation method of existing common ginseng saponin Rh 2, there are many shortcomings, and such as selectivity is low,
Rh2 contents are low cannot to prepare high-purity object, and technique is coarse, be not suitable for amplification production etc..
The content of the invention
Above-mentioned in order to solve the problems, such as, the present invention is mutated wild type glucuroide, and it is big to obtain enzymatic activity
The glucoside enzyme mutant that big raising, ginseng saponin Rh 2 conversion ratio greatly improve.
One of the objects of the present invention is to provide glucoside enzyme mutants.
Another object of the present invention is to provide the application of glucoside enzyme mutant.
Another object of the present invention is to provide the preparation method of the ginseng saponin Rh 2 of glucoside enzyme mutant.
The technical solution used in the present invention is：
A kind of method of Enzyme catalyzed synthesis ginseng saponin Rh 2, using ginseng sapoglycoside Rg 3 as substrate, in urging for glucuroide
Under change, reaction generation ginseng saponin Rh 2；The glucuroide derives from Terrabacter ginsenosidimutans；It urges
The temperature for changing reaction is 25~50 DEG C；
The concentration of ginseng sapoglycoside Rg 3 is 1%~5%w/v in catalystic converter system, and glucoside enzyme dosage is substrate ginseng
0.01~0.06 times of saponin(e Rg3 weight, surplus is for water or phosphate buffer and for dissolving helping for ginseng sapoglycoside Rg 3
Solvent；
The pH value of the catalystic converter system is 6.5~9.5.
Further, the amino acid sequence of the glucuroide as shown in SEQ ID NO 2 or is SEQ ID NO
Amino acid sequence shown in 2 is through obtained by following at least one catastrophe：
The glutamic acid E of the 71st and 72, valine V are sported into threonine T, glutamic acid E respectively；
It is histidine H by the glutamine Q-spoiling of the 92nd；
The serine S of the 109th and 114, proline P are sported into alanine A, glycine G respectively；
The proline P of the 135th is sported into alanine A；
The phenylalanine F of the 168th is sported into tyrosine Y；
It is glutamic acid E by the glutamine Q-spoiling of the 240th；
The lysine K of the 344th is sported into arginine R；
The glutamic acid E of the 412nd and 416, aspartic acid D are sported into aspartic acid D, glutamic acid E respectively；
The tyrosine Y of the 469th is sported into phenylalanine F.
Further, the cosolvent of the ginseng sapoglycoside Rg 3 selected from methanol, ethyl alcohol, 1%~20%v/v DMSO in
It is at least one.
Further, the cosolvent of the ginseng sapoglycoside Rg 3 is the DMSO of 1%~20%v/v.
Further, the time of the catalytic reaction is 4~12h.
Further, the phosphate buffer is 0.08~0.12M phosphate buffers of pH 7.2~7.8.
Further, ginseng sapoglycoside Rg 3 need to be first dissolved in cosolvent, be slow added into glucuroide, and side adds in
It stirs on side.It is slowly added to that the Rg3 being dissolved in cosolvent can be avoided to form thick liquid, can not turn so as to cause stirrer
It is dynamic.In above-mentioned three kinds of cosolvents, minimum with the degree of the such phenomenon of DMSO appearance, other two solvent is easier to occur this existing
As.
Further, the glucuroide is added in the form of enzyme powder or enzyme solution.
Further, the enzyme solution be thalline is broken into born of the same parents with phosphate buffer ultrasonic wave, centrifuging and taking supernatant obtains.
Glucoside enzyme mutant, amino acid sequence is through following at least one catastrophe institute as shown in SEQ ID NO 2
：
The glutamic acid E of the 71st and 72, valine V are sported into threonine T, glutamic acid E respectively；
It is histidine H by the glutamine Q-spoiling of the 92nd；
The serine S of the 109th and 114, proline P are sported into alanine A, glycine G respectively；
The proline P of the 135th is sported into alanine A；
The phenylalanine F of the 168th is sported into tyrosine Y；
It is glutamic acid E by the glutamine Q-spoiling of the 240th；
The lysine K of the 344th is sported into arginine R；
The glutamic acid E of the 412nd and 416, aspartic acid D are sported into aspartic acid D, glutamic acid E respectively；
The tyrosine Y of the 469th is sported into phenylalanine F.
Further, glucoside enzyme mutant nucleotide sequence as shown in SEQ ID NO 1 is prominent through following at least one
Obtained by change situation：
The base sequence GAGGTG of the 211st~216 is sported into ACGGAG；
The base sequence CAG of the 274th~276 is sported into CAC；
Base sequence AGC, CCG of 325th~327 and 340~342 are sported into GCC, GGT respectively；
The base sequence CCG of the 403rd~405 is sported into GGC；
The base sequence TTC of the 502nd~504 is sported into TAC；
The base sequence CAA of the 718th~720 is sported into GAA；
The base sequence CGG of the 1030th~1032 is sported into AAG；
Base sequence GAG, GAT of 1234th~1236 and 1246~1248 are sported into GAC, GAG respectively；
The base sequence TAT of the 1405th~1407 is sported into TTT.
Application of the glucoside enzyme mutant described in any one of the above embodiments in ginseng saponin Rh 2 is prepared.
Further, using ginseng sapoglycoside Rg 3 as substrate in above application.
The beneficial effects of the invention are as follows：
(1) present invention derives from the glucuroide and its mutant enzyme of Terrabacter ginsenosidimutans
The conversion ratio living that Rh2 is changed into substrate Rg3 is up to more than 95.89%, and conversion ratio greatly improves, and reaction selectivity is high, by-product
Few, Rh2 contents are high, available for high-purity Rh2 is prepared, are conducive to further through enzyme process industrialized production ginseng saponin Rh 2.
(2) present invention production ginseng saponin Rh 2 is simple for process, and reaction selectivity is high, and by-product is few, glucose of the present invention
Glycosides enzyme and its mutant are easy to get through Escherichia coli fermentation, and production cost and product quality are suitble to industrialized production better than chemical method.
Reaction condition is mild, belongs to environmentally friendly production technology, meets the environmentally protective industrial standard of country's popularization.
(3) 25~50 DEG C of catalytic reaction temperature, pH value 6.5~9.5, substrate Rg3 concentration are used in the method for the present invention as 1%
~5%w/v, glucoside enzyme dosage are the technical characteristics such as 0.01~0.06 times of Rg3 weight, are more advantageous to Terrabacter
Ginsenosidimutans glucuroides and its mutant enzyme activity improve it and convert Rg3 to the catalytic activity of substrate Rg3
Into the conversion ratio of Rh2, up to more than 95.89%, the generation of by-product is reduced, the purity of gained Rh2 is improved, is more advantageous to enzyme
Method produces the industrialization of ginseng saponin Rh 2.
Specific embodiment
With reference to specific embodiment, the present invention is further illustrated.
1 glucuroide of embodiment and its mutant
By from the beta-glucosidase Rh2-015 of Terrabacter ginsenosidimutans, (gene order is such as
SEQ ID NO:Shown in 1, the protein sequence such as SEQ ID NO of coding:Shown in 2) amino acid sequence through following at least one prominent
Become and obtain glucoside enzyme mutant：
The glutamic acid E of the 71st and 72, valine V are sported into threonine T, glutamic acid E (abbreviation E71T+ respectively
V72E)；
Or/and by the glutamine Q-spoiling of the 92nd be histidine H (abbreviation Q92H)；
Or/and the serine S of the 109th and 114, proline P are sported into alanine A, glycine G (referred to as respectively
S109A+P114G)；
Or/and the proline P of the 135th is sported into alanine A (abbreviation P135A)；
Or/and the phenylalanine F of the 168th is sported into tyrosine Y (abbreviation F168Y)；
Or/and by the glutamine Q-spoiling of the 240th be glutamic acid E (abbreviation Q240E)；
Or/and the lysine K of the 344th is sported into arginine R (abbreviation K344R)；
Or/and the glutamic acid E of the 412nd and 416, aspartic acid D are sported into aspartic acid D, glutamic acid E (letters respectively
Claim E412D+D416E)；
Or/and the tyrosine Y of the 469th is sported into phenylalanine F (abbreviation Y469F).
The preparation of 2 glucoside enzyme mutant of embodiment
Rite-directed mutagenesis is carried out to Rh2-015 by inverse PCR technique, prepares single double-mutant storehouse.
It will be from beta-glucosidase gene Rh2-015 (the gene sequences of Terrabacter ginsenosidimutans
Row such as SEQ ID NO:Shown in 1, the protein sequence such as SEQ ID NO of coding:Shown in 2) it is utilized respectively primer A (SEQ ID NO:
And primer B (SEQ ID NO 3):4) by being handled after PCR amplification acquisition PCR product by digestion, while it is inserted into expression
Nde I and the EcoR I sites of carrier pET22b (+), obtain recombinant plasmid pET22b-Rh2-015.
According to the mutated site described in embodiment 1, in mutated site design reverse primer (mutant primer sequence such as 2 institute of table
Show), target fragment is expanded using upstream and downstream mutant primer, and corresponding mutation is introduced on primer, with recombinant plasmid pET22b-
Rh2-015 carries out inverse PCR as template, and (Plasmid DNA generally used is PCR product after the processing of Dpn I enzymic digestions template
It is extracted from escherichia coli host, due to endogenic dam methylases, the adenine of the sequence has been methylated,
Therefore can be cut off by Dpn I.And with the DNA of the synthesis such as PCR due to not being methylated, it cannot be cut off, eliminate false sun
Property) Escherichia coli Rosetta (de3) is transformed into, picking colony send sequencing after the screening of Amp.After measuring correctly, dashed forward
Become successful recombinant bacterium, carry out induced expression to being mutated successful recombinant bacterium, you can obtain glucoside enzyme mutant.It is above-mentioned anti-
It is to the system of PCR：
TaKaRa EX Taq HS 0.25μL
10×Ex Taq Buffer 5μL
1 μ L of template plasmid pET22b-Rh2-015
dNTP 4μL
1 μ L of mutant primer upstream (table 2)
1 μ L of mutant primer downstream (table 2)
Sterile water is to 50 μ L.
PCR response procedures are：
98℃、2min；98 DEG C, 10s, 55-65 DEG C, 30s, 30 Xun Huans；72℃、7min；72℃、10min.
The mutant primer in 2 mutational site of table
The detection of 3 glucuroide mutant enzyme vigor of embodiment
(1) preparation of the induced expression of wild type beta-glucosidase recombination bacillus coli and its enzyme solution
It will be from beta-glucosidase gene Rh2-015 (the gene sequences of Terrabacter ginsenosidimutans
Row such as SEQ ID NO:Shown in 1, the protein sequence such as SEQ ID NO of coding:Shown in 2) it is utilized respectively primer A (SEQ ID NO:
And primer B (SEQ ID NO 3):4) by being handled after PCR amplification acquisition PCR product by digestion, while it is inserted into expression
Nde I and the EcoR I sites of carrier pET22b (+), obtain recombinant expression pET22b-Rh2-015.After sequence verification,
Recombinant plasmid is transferred to Escherichia coli Rosetta (de3).The recombination bacillus coli of acquisition is seeded in the LB culture mediums of small size
(Amp containing 100 μ g/mL) after 30~37 DEG C are incubated overnight, the LB culture mediums of 1L volumes are transferred to 1~5% inoculum concentration
In (Amp containing 100 μ g/mL), continue at 30~37 DEG C cultivate OD600Reach 0.6~1.0 final concentration of 0.1mM of addition~
The isopropyl-β-D-thiogalactoside (IPTG) of 1mM, thalline were collected by centrifugation after 20~37 DEG C of 10~20h of induced expression.Hair
Yeast-like fungi body is suspended in the phosphate buffer (pH 7.4) of 50~100mM of 4 times of volumes and ultrasonic wave breaks born of the same parents 20min, in centrifuging and taking
Obtain the enzyme solution of Rh2-015 clearly.
(2) wild type beta-glucosidase enzyme activity determination method
Beta-glucosidase Rh2-015 enzyme solutions are reacted using pNP-Glc as substrate, are added in the reaction system of a 3mL
The 150mM pNP-Glc of 1mL, the dilution enzyme solution of 100 μ L, in pH 7.0 and 37 DEG C of reaction certain times, 0.5M sodium carbonate 5min
Terminate reaction.Product p- nitrophenols measures light absorption value increase at 405nm.1 unit enzyme activity is defined as：1 μM of p- of generation per minute
Nitrophenols is 1U.
(3) beta-glucosidase mutant enzyme vigour-testing method
Beta-glucosidase mutant enzyme vigour-testing method will be mutated with wild type beta-glucosidase in embodiment 2
Successful recombinant bacterium carries out induced expression, and derivational expression method is carried out with above-mentioned (1), and using the method identical with above-mentioned (2)
The enzyme activity and its stability (enzyme activity for preserving more than 80%) of wild type and mutant are compared by enzyme activity determination.
Comparative result is as shown in table 3, there it can be seen that the glucosidase activity after present invention mutation greatly improves,
Temperature stabilizing range and pH stability ranges are unaffected or even stability range is wider simultaneously.
The enzyme activity and stability of 3 wild type of table and mutant beta-glucosidase
4 glucoside enzyme mutant of embodiment and wild type catalysis ginseng sapoglycoside Rg 3 synthesis ginseng saponin Rh 2
Method：β-grape of the different saltant types of the wild type through induced expression, the present invention is separately added into different reactor
The enzyme solution of glycosidase, the 4g ginseng sapoglycoside Rg 3s through the dissolving of cosolvent methanol that each group is respectively added slowly to equivalent (are stirred when adding in
Mix) and pure water, make the final concentration 4%w/v, the final concentration of 0.04%w/v of glucuroide of Rg3 in each group reaction system.Instead
It should be reacted under conditions of 40 DEG C of temperature, 300rpm and pH 8.It is extracted reaction solution at regular intervals in reaction process with stream
Dynamic 50~100 times of phase dilution, sample introduction carries out liquid phase analysis after micro porous filtration, detects response situation.Liquid phase detection uses Kinetex
2.6 μm of C18 100A are analytical column, and acetonitrile and water are mobility, and column temperature is room temperature, Detection wavelength 203nm, and flow velocity is
1.0mL/min.React 10 it is small when after, detect the yield of Rh2.The reaction item of wild type and saltant type beta-glucosidase of the present invention
Part is identical.
As a result：Testing result is as shown in table 4, and glucuroide and its mutant enzyme activity are converted into Rh2's to substrate Rg3
For conversion ratio up to more than 95.89%, wherein Q240E generates Rh2 conversion ratios up to 99.68%.
The effect of 4 wild type of table and mutant beta-glucosidase generation Rh2
5 glucoside enzyme mutant of embodiment catalysis ginseng sapoglycoside Rg 3 synthesis ginseng saponin Rh 2
Method：16ml 0.1M phosphate buffers (pH7.5) are added in into reaction kettle to stir simultaneously, add in grape afterwards
Glucosides enzyme mutant (Q240E) 0.04g, stirring in addition, weighing in 4g substrates Rg3 input beakers, add in 20ml DMSO,
400rpm magnetic agitations.It after substrate dissolving, is slowly added in reaction kettle and (stirs while adding), pure water is added to make anti-to 100mL
Substrate in answering is sufficiently mixed with enzyme, 50 DEG C of water-baths, and it is 7-8 that sodium hydroxide, which adjusts pH value,.React 6 it is small when after, detect the production of Rh2
Amount.
As a result：Experimental result is shown, by purifying when reaction 6 is small, obtains Rh2 yield 3.16g, it is seen that saltant type glucoside
Enzyme is 99.68% to the conversion ratio of substrate.
6 glucoside enzyme mutant of embodiment catalysis ginseng sapoglycoside Rg 3 synthesis ginseng saponin Rh 2
Method：Saltant type beta-glucosidase (P135A) and 0.1M phosphate buffers are added in the reactor
(pH7.5), the ginseng sapoglycoside Rg 3 (stirring while adding) dissolved through cosolvent methanol is slow added into, makes the final concentration of Rg3
4%w/v, the concentration of glucuroide is 0.12%w/v.Reaction carries out under conditions of 25 DEG C of temperature, 400rpm and pH 9.5
Reaction.React 12 it is small when after, detect the yield of Rh2, saltant type glucuroide is 99.05% to the conversion ratio of substrate.
7 glucoside enzyme mutant of embodiment catalysis ginseng sapoglycoside Rg 3 synthesis ginseng saponin Rh 2
Method：Saltant type beta-glucosidase (E412D+D416E) and 0.12M phosphate buffers are added in the reactor
(pH7.8), the ginseng sapoglycoside Rg 3 (stirring while adding) dissolved through cosolvent ethyl alcohol is slow added into, makes the final concentration of Rg3
1%w/v, the concentration of glucuroide is 0.06%w/v.Reaction carries out under conditions of temperature 50 C, 200rpm and pH 6.5
Reaction.React 4 it is small when after, detect the yield of Rh2, saltant type glucuroide is 99.37% to the conversion ratio of substrate.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention and from above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Thailand of nation bioengineering（Shenzhen）Co., Ltd
Green bio synthesis ecoindustrial park Development Co., Ltd of Jiangxi Thailand of nation
<120>A kind of method of Enzyme catalyzed synthesis ginseng saponin Rh 2
<130>
<160> 22
<170> PatentIn version 3.5
<210> 1
<211> 1944
<212> DNA
<213>Artificial sequence
<400> 1
atgaccatga tcaccccgag ctacctgggc gagaccattg aatatagcag cctgcacgcg 60
tgccgtagca ccctggaaga tccgaccccg agccgtcgta aggctccgcc ggttcgtccg 120
gagctgcaca ccaccgaaga cggtgtggcg taccgtgatc tgaacggcaa cggtcgtatg 180
gacccgtatg aggatccgcg tctgccggtt gaggtgcgcg ttgaagacct gctgggccgt 240
ctgagcctgg aggaaaaagt tggtctgatg ttccagaccg tgatcgaggc gggcagcgat 300
ggtaccgttc tggaacaccc gggtagcatt agcaagagcc cgaccagcac cgtggttctg 360
gacaaacacc tgacccactt taacgtgcac gcgctggacg atccgcgtat ggcggcgcgt 420
tggagcaacg cgctgcaagc gctggcggaa cgtaccccgc acggtatccc ggtgaccgtt 480
agcaccgacc cgcgtcacgc gttcattgag aacgtgggcg ttagcttcag cgcgggtgcg 540
tttagccagt ggccggaacc gctgggtctg gcggcgctgc gtgacgcgga tgcggttcgt 600
cgttttgcgg atatcgcgcg tcaagagtac gtggcggttg gtattcgtgc ggcgctgcac 660
ccgaccctgg acctggcgac cgaaccgcgt tgggcgcgtc aggcgggtac cttcggtcaa 720
gacccggatc tggtgaccga gctgggtgtt gcgtatctga agggctttca gggtgatagc 780
ctgggcagcg gtagcgttgc gtgcaccagc aagcacttcc cgggtggcgg tccgcagaaa 840
gacggcgagg atgcgcactt tccgtacggt cgtgaacaag tgtatccggg cggtcgtttc 900
gcggaccacc tgaaaccgtt tccgccgatc attgaggcgg gtaccgcggg catcatgccg 960
tactatggca tgccggttga cctggtggtt gatggtgtgg agatcgaacc gattggcttc 1020
ggttacaaca agcaggtggt taccggcctg ctgcgtgaga aactgggcta tgacggtgtg 1080
gttgtgaccg attgggaact ggttaacgac aaccacgtgg gtgatcaagt tctgccggcg 1140
cgtgcgtggg gcgtggaaca cctggacccg cacggtcgta tggagctgat cctggaagcg 1200
ggtgcggacc agttcggcgg tgaagaatgc gttgagattc tgctggatct ggtggcgcaa 1260
ggtcgtgtta ccgaggcgcg tgtggacgaa agcgcgcgtc gtatcctggc ggtgaagttc 1320
cgtctgggtc tgtttgaaaa cccgtacgtt gacgaggatg aagcggcggc gaccgtgggt 1380
cgtgacgatt ttcgtgagga aggttatgcg gcgcaggcgc gtagcgtgac cgttctgcac 1440
cacgagggcg gtcgtctgcc gctggaacac ggcctgcgta tttacgcgga gcaagttagc 1500
ccggaagcgg tggcgcgtca cggtaaactg gttgatcgtc cggaggacgc ggatgtggcg 1560
gttgtgcgtc tgaccgcgcc gttcgacccg cgtagcgacc tgttcctgga aagctggttt 1620
caccagggca gcctggactt tccgccgggt ctggttgcgc gtctggagcg tatcgcggcg 1680
gtgtgcccgc tggttgtgga cgttgtgctg gatcgtccgg cggttctgac cccgctgctg 1740
cgtttcgcga gcgcggttgt gggcagcttt ggtagctgcg acgatgcgct gctggacgcg 1800
ctgaccggta ccattgcgcc ggtgggtcgt ctgccgttcg acctgccgcg tagcatggat 1860
caagttcgtg cgcacggcga agatgtgccg ggttacgacg atccgctgtt cccgtttggc 1920
cacggtctgc gtctggacac cgag 1944
<210> 2
<211> 648
<212> PRT
<213>Artificial sequence
<400> 2
Met Thr Met Ile Thr Pro Ser Tyr Leu Gly Glu Thr Ile Glu Tyr Ser
1 5 10 15
Ser Leu His Ala Cys Arg Ser Thr Leu Glu Asp Pro Thr Pro Ser Arg
20 25 30
Arg Lys Ala Pro Pro Val Arg Pro Glu Leu His Thr Thr Glu Asp Gly
35 40 45
Val Ala Tyr Arg Asp Leu Asn Gly Asn Gly Arg Met Asp Pro Tyr Glu
50 55 60
Asp Pro Arg Leu Pro Val Glu Val Arg Val Glu Asp Leu Leu Gly Arg
65 70 75 80
Leu Ser Leu Glu Glu Lys Val Gly Leu Met Phe Gln Thr Val Ile Glu
85 90 95
Ala Gly Ser Asp Gly Thr Val Leu Glu His Pro Gly Ser Ile Ser Lys
100 105 110
Ser Pro Thr Ser Thr Val Val Leu Asp Lys His Leu Thr His Phe Asn
115 120 125
Val His Ala Leu Asp Asp Pro Arg Met Ala Ala Arg Trp Ser Asn Ala
130 135 140
Leu Gln Ala Leu Ala Glu Arg Thr Pro His Gly Ile Pro Val Thr Val
145 150 155 160
Ser Thr Asp Pro Arg His Ala Phe Ile Glu Asn Val Gly Val Ser Phe
165 170 175
Ser Ala Gly Ala Phe Ser Gln Trp Pro Glu Pro Leu Gly Leu Ala Ala
180 185 190
Leu Arg Asp Ala Asp Ala Val Arg Arg Phe Ala Asp Ile Ala Arg Gln
195 200 205
Glu Tyr Val Ala Val Gly Ile Arg Ala Ala Leu His Pro Thr Leu Asp
210 215 220
Leu Ala Thr Glu Pro Arg Trp Ala Arg Gln Ala Gly Thr Phe Gly Gln
225 230 235 240
Asp Pro Asp Leu Val Thr Glu Leu Gly Val Ala Tyr Leu Lys Gly Phe
245 250 255
Gln Gly Asp Ser Leu Gly Ser Gly Ser Val Ala Cys Thr Ser Lys His
260 265 270
Phe Pro Gly Gly Gly Pro Gln Lys Asp Gly Glu Asp Ala His Phe Pro
275 280 285
Tyr Gly Arg Glu Gln Val Tyr Pro Gly Gly Arg Phe Ala Asp His Leu
290 295 300
Lys Pro Phe Pro Pro Ile Ile Glu Ala Gly Thr Ala Gly Ile Met Pro
305 310 315 320
Tyr Tyr Gly Met Pro Val Asp Leu Val Val Asp Gly Val Glu Ile Glu
325 330 335
Pro Ile Gly Phe Gly Tyr Asn Lys Gln Val Val Thr Gly Leu Leu Arg
340 345 350
Glu Lys Leu Gly Tyr Asp Gly Val Val Val Thr Asp Trp Glu Leu Val
355 360 365
Asn Asp Asn His Val Gly Asp Gln Val Leu Pro Ala Arg Ala Trp Gly
370 375 380
Val Glu His Leu Asp Pro His Gly Arg Met Glu Leu Ile Leu Glu Ala
385 390 395 400
Gly Ala Asp Gln Phe Gly Gly Glu Glu Cys Val Glu Ile Leu Leu Asp
405 410 415
Leu Val Ala Gln Gly Arg Val Thr Glu Ala Arg Val Asp Glu Ser Ala
420 425 430
Arg Arg Ile Leu Ala Val Lys Phe Arg Leu Gly Leu Phe Glu Asn Pro
435 440 445
Tyr Val Asp Glu Asp Glu Ala Ala Ala Thr Val Gly Arg Asp Asp Phe
450 455 460
Arg Glu Glu Gly Tyr Ala Ala Gln Ala Arg Ser Val Thr Val Leu His
465 470 475 480
His Glu Gly Gly Arg Leu Pro Leu Glu His Gly Leu Arg Ile Tyr Ala
485 490 495
Glu Gln Val Ser Pro Glu Ala Val Ala Arg His Gly Lys Leu Val Asp
500 505 510
Arg Pro Glu Asp Ala Asp Val Ala Val Val Arg Leu Thr Ala Pro Phe
515 520 525
Asp Pro Arg Ser Asp Leu Phe Leu Glu Ser Trp Phe His Gln Gly Ser
530 535 540
Leu Asp Phe Pro Pro Gly Leu Val Ala Arg Leu Glu Arg Ile Ala Ala
545 550 555 560
Val Cys Pro Leu Val Val Asp Val Val Leu Asp Arg Pro Ala Val Leu
565 570 575
Thr Pro Leu Leu Arg Phe Ala Ser Ala Val Val Gly Ser Phe Gly Ser
580 585 590
Cys Asp Asp Ala Leu Leu Asp Ala Leu Thr Gly Thr Ile Ala Pro Val
595 600 605
Gly Arg Leu Pro Phe Asp Leu Pro Arg Ser Met Asp Gln Val Arg Ala
610 615 620
His Gly Glu Asp Val Pro Gly Tyr Asp Asp Pro Leu Phe Pro Phe Gly
625 630 635 640
His Gly Leu Arg Leu Asp Thr Glu
645
<210> 3
<211> 29
<212> DNA
<213>Artificial sequence
<400> 3
cgccatatga tgaccatgat caccccgag 29
<210> 4
<211> 29
<212> DNA
<213>Artificial sequence
<400> 4
ccggaattcc tcggtgtcca gacgcagac 29
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence
<400> 5
ctgccggtta cggagcgcg 19
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
gctccgtaac cggcagacgc g 21
<210> 7
<211> 21
<212> DNA
<213>Artificial sequence
<400> 7
ggtctgatgt tccacaccgt g 21
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence
<400> 8
cgcctcgatc acggtgtgga 20
<210> 9
<211> 26
<212> DNA
<213>Artificial sequence
<400> 9
cgggtgccat tagcaagagc ggtacc 26
<210> 10
<211> 25
<212> DNA
<213>Artificial sequence
<400> 10
ctggtaccgc tcttgctaat ggcac 25
<210> 11
<211> 21
<212> DNA
<213>Artificial sequence
<400> 11
ctggacgatg gccgtatggc g 21
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> 12
tacgggcatc gtccagcgcg 20
<210> 13
<211> 19
<212> DNA
<213>Artificial sequence
<400> 13
ccgcgtcacg cgtacattg 19
<210> 14
<211> 23
<212> DNA
<213>Artificial sequence
<400> 14
cccacgttct caatgtacgc gtg 23
<210> 15
<211> 24
<212> DNA
<213>Artificial sequence
<400> 15
cggtgaagac ccggatctgg tgac 24
<210> 16
<211> 24
<212> DNA
<213>Artificial sequence
<400> 16
gggtcttcac cgaaggtacc cgcc 24
<210> 17
<211> 25
<212> DNA
<213>Artificial sequence
<400> 17
ggttacaacc ggcaggtggt taccg 25
<210> 18
<211> 22
<212> DNA
<213>Artificial sequence
<400> 18
gccggtaacc acctgccggt tg 22
<210> 19
<211> 24
<212> DNA
<213>Artificial sequence
<400> 19
gcgttgacat tctgctggag ctgg 24
<210> 20
<211> 22
<212> DNA
<213>Artificial sequence
<400> 20
ccagctccag cagaatgtca ac 22
<210> 21
<211> 23
<212> DNA
<213>Artificial sequence
<400> 21
ggaaggtttt gcggcgcagg cgc 23
<210> 22
<211> 24
<212> DNA
<213>Artificial sequence
<400> 22
ctgcgccgca aaaccttcct cacg 24
Claims (10)
- A kind of 1. method of Enzyme catalyzed synthesis ginseng saponin Rh 2, it is characterised in that：Using ginseng sapoglycoside Rg 3 as substrate, in glucose Under the catalysis of glycosides enzyme, reaction generation ginseng saponin Rh 2；The glucuroide derives from Terrabacter ginsenosidimutans；The temperature of catalytic reaction is 25~50 DEG C；The concentration of ginseng sapoglycoside Rg 3 is 1%~5%w/v in catalystic converter system, and glucoside enzyme dosage is substrate ginsenoside 0.01~0.06 times of Rg3 weight, surplus are water or phosphate buffer and the cosolvent for dissolving ginseng sapoglycoside Rg 3；The pH value of the catalystic converter system is 6.5~9.5.
- 2. the according to the method described in claim 1, it is characterized in that, amino acid sequence of the glucuroide such as SEQ ID Shown in NO 2 or it is amino acid sequence shown in SEQ ID NO 2 through obtained by following at least one catastrophe：The glutamic acid E of the 71st and 72, valine V are sported into threonine T, glutamic acid E respectively；It is histidine H by the glutamine Q-spoiling of the 92nd；The serine S of the 109th and 114, proline P are sported into alanine A, glycine G respectively；The proline P of the 135th is sported into alanine A；The phenylalanine F of the 168th is sported into tyrosine Y；It is glutamic acid E by the glutamine Q-spoiling of the 240th；The lysine K of the 344th is sported into arginine R；The glutamic acid E of the 412nd and 416, aspartic acid D are sported into aspartic acid D, glutamic acid E respectively；The tyrosine Y of the 469th is sported into phenylalanine F.
- 3. according to the method described in claim 1, it is characterized in that, the cosolvent of the ginseng sapoglycoside Rg 3 is selected from methanol, second At least one of the DMSO of alcohol, 1%~20%v/v.
- 4. according to the method described in claim 1, it is characterized in that, the time of the catalytic reaction is 4~12h.
- 5. according to the method described in claim 1, it is characterized in that, the phosphate buffer is the 0.08 of pH 7.2~7.8 ~0.12M phosphate buffers.
- 6. according to the method described in claim 1, it is characterized in that, the ginseng sapoglycoside Rg 3 need to first be dissolved in the cosolvent In, it is slow added into the glucuroide, stirs while adding.
- 7. glucoside enzyme mutant, which is characterized in that amino acid sequence is through following at least one prominent as shown in SEQ ID NO 2 Obtained by change situation：The glutamic acid E of the 71st and 72, valine V are sported into threonine T, glutamic acid E respectively；It is histidine H by the glutamine Q-spoiling of the 92nd；The serine S of the 109th and 114, proline P are sported into alanine A, glycine G respectively；The proline P of the 135th is sported into alanine A；The phenylalanine F of the 168th is sported into tyrosine Y；It is glutamic acid E by the glutamine Q-spoiling of the 240th；The lysine K of the 344th is sported into arginine R；The glutamic acid E of the 412nd and 416, aspartic acid D are sported into aspartic acid D, glutamic acid E respectively；The tyrosine Y of the 469th is sported into phenylalanine F.
- 8. glucoside enzyme mutant according to claim 7, which is characterized in that the nucleotide as shown in SEQ ID NO 1 Sequence is through obtained by following at least one catastrophe：The base sequence GAGGTG of the 211st~216 is sported into ACGGAG；The base sequence CAG of the 274th~276 is sported into CAC；Base sequence AGC, CCG of 325th~327 and 340~342 are sported into GCC, GGT respectively；The base sequence CCG of the 403rd~405 is sported into GGC；The base sequence TTC of the 502nd~504 is sported into TAC；The base sequence CAA of the 718th~720 is sported into GAA；The base sequence CGG of the 1030th~1032 is sported into AAG；Base sequence GAG, GAT of 1234th~1236 and 1246~1248 are sported into GAC, GAG respectively；The base sequence TAT of the 1405th~1407 is sported into TTT.
- 9. application of the claim 7~8 any one of them glucoside enzyme mutant in ginseng saponin Rh 2 is prepared.
- 10. application according to claim 9, which is characterized in that using ginseng sapoglycoside Rg 3 as substrate.
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| WO2020215265A1 (en) * | 2019-04-25 | 2020-10-29 | 邦泰生物工程(深圳)有限公司 | Method for preparing mixture of rare ginsenosides rh3 and rk2 and mixture thereof |
| CN112384233A (en) * | 2019-01-31 | 2021-02-19 | 邦泰生物工程(深圳)有限公司 | A method for preparing radix Codonopsis extract from radix Codonopsis leftover |
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| CN110791490B (en) * | 2019-11-15 | 2021-03-30 | 东北师范大学 | Recombinant beta-glucosidase CB-3B and application thereof in production of ginsenoside Rg3 |
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| CN102782125A (en) * | 2009-10-23 | 2012-11-14 | 韩国生命工学研究院 | Novel ginsenoside glycosidase derived from the genus terrabacter, and use thereof |
| KR101340079B1 (en) * | 2012-09-03 | 2014-01-24 | 한국과학기술원 | Biotransformation of ppt-type ginsenoside using terrabacter sp.-derived b-glucosidase |
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| CN103571809B (en) * | 2012-07-24 | 2015-06-03 | 中国科学院上海生命科学研究院 | Novel beta-glucosidase, coding gene thereof and applications thereof |
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| CN102782125A (en) * | 2009-10-23 | 2012-11-14 | 韩国生命工学研究院 | Novel ginsenoside glycosidase derived from the genus terrabacter, and use thereof |
| KR101340079B1 (en) * | 2012-09-03 | 2014-01-24 | 한국과학기술원 | Biotransformation of ppt-type ginsenoside using terrabacter sp.-derived b-glucosidase |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112384233A (en) * | 2019-01-31 | 2021-02-19 | 邦泰生物工程(深圳)有限公司 | A method for preparing radix Codonopsis extract from radix Codonopsis leftover |
| CN112384233B (en) * | 2019-01-31 | 2022-07-26 | 邦泰生物工程(深圳)有限公司 | A method for preparing radix Codonopsis extract from radix Codonopsis leftover |
| WO2020215265A1 (en) * | 2019-04-25 | 2020-10-29 | 邦泰生物工程(深圳)有限公司 | Method for preparing mixture of rare ginsenosides rh3 and rk2 and mixture thereof |
| CN112313342A (en) * | 2019-04-25 | 2021-02-02 | 邦泰生物工程(深圳)有限公司 | Preparation method of rare ginsenoside Rh3 and Rk2 mixture and mixture thereof |
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| CN108064309B (en) | 2021-07-20 |
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| CN109837261A (en) | 2019-06-04 |
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